I. Field of the Invention
The present invention relates generally to the fields of molecular biology and virology. More particularly, it concerns an infectious clone of GBV-C, which can be used in treatment of other related hepatitis viruses infections and HIV, as well as broader uses in therapeutic and preventative therapies.
II. Description of Related Art
A. GB Virus Type C
Hepatitis C virus (HCV) is responsible for causing hepatitis C, a disease that chronically affects approximately four million Americans, many of whom may develop liver disease. Hepatitis C annually accounts for as many as 1,000 liver transplants in the United States and between 8,000 and 10,000 deaths. There is no vaccine or preventative treatment against HCV infection and treatment regimens may cause unwanted side effects.
GB virus type C (GBV-C), also known as hepatitis G virus (HGV), is a recently described virus whose genomic organization and nucleotide sequence places it in the Flavivirus family (Robertson et al., 1998). It is the most closely related human virus to HCV (Leary et al., 1996; Linnen et al., 1996; Simons et al., 1995), a major, worldwide pathogen (Robertson et al., 1998). It has been suggested that these viruses should be classified together with non-human GB-hepatitis agents as the hepacivirus genus. Both GBV-C and HCV appear to utilize the LDL receptor for viral entry (Agnello et al., 1999). Thus, comparison of GBV-C and HCV may provide insight into the reasons why HCV does not appear to replicate as efficiently in cell culture as GBV-C, and why GBV-C is cleared more efficiently by the host immune response than HCV (Gutierrez et al., 1997; Thomas et al., 1998; Toyoda et al., 2000).
Although GBV-C was originally associated with post-transfusion hepatitis in humans (Linnen et al., 1996), subsequent epidemiological studies indicated that it does not cause acute or chronic hepatitis (Alter et al., 1997a; Alter et al., 1997b). In addition, experimental GBV-C infection of chimpanzees was not associated with acute hepatitis (Bukh et al., 1998).
Persistent GBV-C viremia (as detected by RT-PCR) is common, with 0.9% to 3% of healthy U.S. blood donors and approximately 20%-30% of patients with HCV infection persistently infected with GBV-C (Dawson et al., 1996; Feucht et al., 1997; Guitierrez et al., 1997; Simons et al., 1995a; Simons et al., 1995b; Tacke et al., 1997). Following infection, about 80% of people clear their viremia, concomitantly developing antibody to the GBV-C E2 protein (Feucht et al., 1997; Gutierrez et al., 1997; Thomas et al., 1998). Thus, it is estimated that approximately 20% of infected people remain viremic for long periods of time. GBV-C appears to be transmitted primarily by parenteral exposure (Simons et al., 1995), although there are data suggesting that sexual and/or household transmission of GBV-C infection may occur (Akiyoshi et al., 1999; de Martino et al., 1998; Nerurkar et al., 1998; Tanaka et al., 1997; Wu et al., 1997).
B. GBV-C and HIV
Recently, GBV-C has been investigated in the context of HIV infection. The course of HIV-1 infection is extremely variable among infected individuals, although the reasons for this observation are incompletely understood. Individuals whose HIV disease progresses slowly are often called long-term non-progressors (LTNPs). The prevalence of LTNPs varies from 1% to 25% of infected people, depending upon the definition used (reviewed in Easterbrook, 1999). There are no specific clinical criteria for LTNP; however, non-progression generally implies the absence of HIV-related clinical disease 10 or more years following infection and an absolute CD4 count of ≧500 cells/mm3 (Easterbrook, 1999). Evaluation of LTNP's has identified HIV isolates with deletions in key replicative genes (Deacon et al., 1995) and host genetic factors, including specific HLA haplotypes (reviewed in reference Rowland-Jones, 1999) or, in some individuals, polymorphisms that result in absent or reduced expression of HIV co-receptors (Huang et al., 1996). However, these findings are uncommon and thought to account for no more than one-third of LTNP's (Rowland-Jones, 1999).
Persistent GBV-C infection is common in humans, with infection rates of approximately 1.8% in healthy blood donors, 15% in HCV positive people (Dawson et al., 1996), and 35%-40% in HIV positive individuals. GBV-C infection can persist for decades in the absence of any clinical morbidity or mortality. Among immune-competent individuals, it is estimated that 60% to 75% of GBV-C-infected people clear the infection, concomitantly developing antibodies to the envelope glycoprotein E2 (Thomas et al., 1998). GBV-C has been propagated in cultures of peripheral blood mononuclear cells (PBMC's) (Fogeda et al., 1999).
In 1998, Toyoda et al. found that hemophiliacs co-infected with HIV and GBV-C (also known as hepatitis G virus, HGV, or GBVC-HGV) had a lower plasma HIV RNA concentration and a lower incidence of AIDS diagnoses compared to those infected with HIV alone (Toyoda et al., 1998), although the differences were not statistically significant. In contrast, Sabin and colleagues found an increased rate of AIDS and death in hemophiliacs “exposed” to GBV-C (Sabin et al., 1998) compared to non-exposed individuals. This study included HIV-positive subjects who were either GBV-C viremic as determined by detection of GBV-C RNA in plasma, or HIV-infected people who were not viremic but were anti-GBV-C E2 antibody-positive. Although the mortality rate was higher among the GBV-C “exposed” individuals, the results were not statistically significant. Looking at HIV-infected persons, Lefrere and colleagues reported a significant delay in the rate of CD4+ T cell decline, development of AIDS, and death in 23 HIV-positive individuals with GBV-C viremia compared to 72 HIV-infected people without GBV-C viremia (Lefrère et al., 1999). In this study, HIV-infected individuals who were also GBV-C-positive were compared to HIV-infected individuals who were GBV-C-negative. When these subjects were matched by age, sex, baseline HIV RNA load, and baseline CD4 T cell count, HIV disease progression appeared to be worse in GBV-C-negative subjects.
Human herpesvirus 6 suppresses HIV replication in CD4+ T cells and dendritic cells (Asada et al., 1999), and recently, Pinto et al. demonstrated induction of in vitro anti-HIV activity by influenza virus (Pinto et al., 2000). Pinto and colleagues demonstrated that this anti-HIV activity was inhibited in part by anti-interferon alpha (γ-IFN) antibodies. GBV-C and its close relative HCV are unusual among human RNA viruses in that they cause persistent infection without a DNA intermediate or known latent stage in their replication cycle. Although the mechanism by which GBV-C is able to persist in vivo is unknown, there are some suggestive data potentially explaining HCV persistence. Two HCV proteins (the envelope glycoprotein E2 and the nonstructural protein NS5a) interact and inhibit an interferon-induced, RNA dependent protein kinase (PKR) (Gale et al., 1998; Taylor et al., 1999). PKR is one of several enzymes induced by γ-IFN, and one of the activities of PKR is to inhibit viral protein synthesis. Since GBV-C and HCV contain numerous predicted stem-loop structures in their positive sense RNA genomes, and replicate via a negative sense RNA intermediate, both viruses would potentially induce -IFN in PBMC cultures by presenting double stranded RNA in the cytoplasm of the cell. There appears to be an animal model supportive of this hypothesis. Two reports suggest that bovine viral diarrhea virus (BVDV; a flavivirus related to GBV-C) induces γ-IFN in cattle (Rinaldo, et al., 1976), and BVDV infection ameliorates experimental bovine respiratory syncytial virus infection in calves (Elvander et al., 1998).
During progressive human immunodeficiency virus type 1 (HIV-1) infection, the virus-specific immune responses of an infected subject gradually deteriorate, leading to the development of acquired immunodeficiency syndrome (AIDS). Most infected patients do not exhibit overt clinical manifestations of the disease for six to ten years following initial infection, however, most individuals infected with HIV eventually die from conditions or infections that the individual's immune system is no longer equipped to fight. While treatment for AIDS has been forthcoming, no effective cure has been reported. Thus, preventative and treatment options against HIV infection and the development of AIDS remain highly desirable. Use of GBV-C is advantageous because of the relative innocuousness of the virus.
C. Infectious Nucleic Acids
Full length cDNAs or RNA transcripts of several RNA viruses including hepatitis A virus, GBV-B, and HCV are infectious in cell culture or animal inoculation studies (Beard et al., 1999; Bukh et al., 1999; Cohen et al., 1987; Emerson et al., 1992; Hong et al., 1999; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1998). These infectious clones are useful for genetic studies and allow a precise method for evaluating evolution of viruses that normally exist in molecular quasispecies. Although several infectious HCV clones have been described, all of these rely upon inoculation of transcribed RNA into susceptible primate species, and none were shown to be infectious in vitro (Beard et al., 1999; Hong et al., 1999; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1998). Thus, these HCV infectious clones have only limited application (Beard et al., 1999; Hong et al., 1999; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1998). As the most closely related human flavivirus to HCV, the construction of an infectious GBV-C cDNA clone that replicates in vitro would provide an important tool for studying human hepacivirus replication.